Monoclonal antibody technologies have generated important tools for cancer diagnoses, therapeutics and prognoses. Generation of murine monoclonal antibodies with hybridoma technology, phage display, or other technologies is especially critical for both basic and clinical sciences. Anticancer monoclonal antibodies used in clinical applications, e.g. Herceptin and C225, were originally produced from mouse. They are beneficial to patients with solid tumors such as breast cancer, ovarian cancer, and head/neck cancers.
Much research has been done to identify antibodies that bind to surface molecules of cancer cells by whole-cell immunization followed by antibody screening. Although the theory of this approach is very attractive, not many therapeutic antibodies have been found after years of effort. This approach has proved difficult for several reasons.
One reason is that standard immunization procedures frequently fail to provide monoclonal antibodies with desired specificity because of the different immunogenicity of various antigen epitopes on the cells used for immunization. The immune response in favor of the more immunogenic epitopes or proteins is dominant. Traditional immunization usually results in generation of monoclonal antibodies to limited epitopes and immunodominant molecules.
Another difficulty with whole-cell immunization is that whole-cell immunization provides an efficient antigen concentration for limited surface molecules that have high antigen density, but a much lower effective antigen concentration for other surface molecules. Because of the complexity of surface antigens, traditional whole-cell immunization and screening may not produce a broad range of antibody specificities.
Also, cancer cells share many common surface antigens with normal cells, including red blood cells and white blood cells in the circulation. The immune response from a mouse may therefore not be tumor-specific although cancer cells are used as an immunogen. Phage display technology is a powerful tool to select tumor-specific antibodies. However, it is a challenge to select tumor-specific antibodies from animals such as mouse, rabbits, and chickens immunized with human cancer cells. In particular, antibodies against common antigens on both normal cells and cancer cells could severely interfere with screening for therapeutic anticancer antibodies, significantly decreasing the success of the whole cell panning approach. In fact, antisera from mice immunized with seven different cancer cell lines has been found to cross-react with human red blood cells (RBC). See Table 1. Therefore, screening for tumor-specific antibodies can be time-consuming and unproductive.
TABLE 1FACS Analyses of Cross-reactivity of Anticancer Sera to HumanBlood CellsFACS withOriginalFACS withCancer CellsRBC(Post-bleed/(Post-bleed/CancerAnimalPre-bleed)Pre-bleed)Cell LinesTypeNumberGeo-MeanGeo-MeanMDA-MB-435Breast5350 X149 XMCF-7Breast5300 X329 XSK-OV3Ovarian5178 X423 XPC3Prostate4400 X516 XDu145Prostate5420 X661 XKM12L4aColon4300 X307 XA431Head and3275 X557 XNeckCaki-1Renal3300 X160 X
Subtractive immunization has been used to solve the problems described above. Subtractive immunization utilizes a distinct immune tolerization approach that can enhance the generation of monoclonal antibodies to desired antigens. Subtractive immunization is based on tolerizing the host animal to immunodominant or otherwise undesired antigens that may be structurally or functionally related to the antigens of interest. Tolerization of the host animal has been achieved through one of three methods: high zone, neonatal, or drug-induced tolerization. The tolerized animal is inoculated with the desired antigens and antibodies are generated in the subsequent immune response and are then screened for the desired reactivity. However, recent study suggested that neonatal “tolerization” induces immune deviation, not tolerance in the immunological sense. Neonates are not immune-privileged but generate TH2 or TH1 responses, depending on the mode of immunization. Chemical immunosuppression with cyclophosphamide has been the most effective subtractive immunization technique. As those skilled in the art will appreciate, normal cell immunization followed by cyclophosphamide treatment will kill all the proliferating immune cells reactive with normal cell antigens. However, this regimen also kills all of the helper T-cells required for B-cell maturation and differentiation. Therefore, when this regimen is followed by cancer cell immunization to elicit antibodies specific to tumor antigens, only low affinity antibodies of IgM isotype are produced.
It would be advantageous to have improved methods for identifying antibodies which bind to surface molecules of cancer cells but not to normal cells Improved methods for treating individuals suffering from cancer are also desirable.